cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin promoter in vitro.

Usages de l'eau à Gaya (Niger) : entre fortes potentialités et contraintes majeures

Résumé: Le département de Gaya, cadre de notre étude, est situé au sud-ouest de la république du Niger. Il dispose d'un important potentiel hydrique composé des eaux de surface (une centaine de mares permanentes, le fleuve Niger sur 106 km) et de sept aquifères superposés comprenant des nappes de subsurface (affleurantes par endroit) et des nappes artésiennes. L'étude sur les usages de l'eau à Gaya a été menée à travers plusieurs axes centrés sur l'estimation et la répartition spatiale des ressources en eau, le cadre juridique et institutionnel régulant leur mise en valeur, les différents secteurs d'utilisation de l'eau ainsi que les contraintes affectant cette utilisation. L'usage de la cartographie à travers les SIG dans le traitement et l'analyse des données, couplée à notre expérience d'une dizaine d'année de travaux dans la région, a permis de dresser des synthèses richement illustrées permettant de mieux comprendre tous les enjeux liés à la problématique des usages de l'eau dans cette partie du Niger. Contrairement à la vision que l'on a traditionnellement du Sahel où le manque d'eau constitue une des contraintes majeures au développement, ici des conditions locales particulières contredisent ce cliché et transposent le débat sur un autre plan. Il s'agit de la maîtrise de l'eau au niveau local à travers l'élaboration d'une politique appropriée qui tienne compte non seulement des spécificités locales de la ressource, mais aussi des différents types d'usages. La politique de l'eau au Niger, définie selon le Schéma directeur de mise en valeur et de gestion des ressources en eau, à travers la mise en place d'un important arsenal juridique et institutionnel, a eu le mérite de tracer un canevas sur la question, mais a montré ses limites au niveau pratique après dix ans d'essai. En effet au niveau de Gaya, ni l'Etat ni les partenaires au développement (bailleurs de fonds extérieurs) n'ont tenu compte des caractéristiques locales de la ressource ou du contexte socioéconomique particulier de la région. Ce qui a entraîné la réalisation d'infrastructures inadaptées aux réalités hydrogéologiques locales ainsi que des choix inappropriés au niveau de certains aménagements. En dépit de l'abondance de la ressource, son accès tant au niveau quantitatif que qualitatif, reste difficile pour une grande partie des acteurs ruraux. Les différents handicaps rencontrés dans la mise en valeur des ressources en eau résultent de cette incohérence de la politique nationale de l'eau, mais aussi de la difficulté de son application sur le terrain où persiste un pluralisme juridique caractérisé par la cohabitation de deux systèmes de régulation à savoir les droits coutumiers et la législation moderne. Ces différents éléments mis en évidence dans cette étude sur la zone de Gaya pourraient servir de base pour un meilleur aménagement des ressources en eau dans le cadre plus large d'une politique d'aménagement du territoire prenant en compte tous les facteurs tant physiques que socioéconomiques de la région. Abstract: The department of Gaya, in which this study was done, is located in the SW area of the Republic of Niger. It has an important hydrological potential composed of surface water (approximately 100 permanent ponds, 106 km of the Niger River) and 7 bodies of underground water sources including sub-surface and artresan wells. This study of the exploitation of wtaer in Gaya has been carried out employing several parameters based on: the estimation and spacial distribution of water ressources, the juridic and institutional rules governing their utilisation and the various constraints affecting this exploitation. The use of mapping when treating and analysing data, coupled with ten years personel field experience, resulted in a richly illustrated synthesis of this data. This, in turn, led to a better comprehension of all the factors related to problems of water utilisation in this particular region of Niger. Contrary to the generally accepted view that the lack of water ressources is a major contributing factor to the lack of development in the Sahel, in Gaya the local conditions contradict this statement. In this region, and at the local level, the proper use of water is based on the elaboration of an appropriate policy which takes into account not only the local specifics of water ressources but the various types of water utilsation as well. Local use of water and water ressources are dependant on established rules. Water policy in Niger is defined by the General Schema based on an important institutional and judicary arsenal of rules and regulations. However, after a ten-year trial period, this system was shown to have its limitations. In Gaya, neither the State nor the development agencies took into consideration local characteristics nor the socio-economic context of the region. This, in turn, resulted in putting in place infrastructures that were not adapted to local hydrogeological realities as well as inappropriate choices in land planning and development. In spite of the abundance of water ressources, access to them remains difficult for most of the rural population. The various difficulties encountered are the result of incoherent water policies on a national level as well as the lack of practical application in this area. This is due to a double judicary system where two regulatory systems co-exist:traditional laws and modern legislation. the different elements brought out by this study could serve as a basis for a better utilisation of water ressources on a larger scale in which land planning and development policies would take into consideration all the physcial as well as the socio-economical factors of this region.

Key elements for, and indicators of, a successful transition: an international Delphi study.

PURPOSE: The purpose of this study was to reach an international consensus to determine what key elements should be part of a transition program and what indicators could be used to assess its success. METHODS: For this purpose, a Delphi study including an international panel of 37 experts was carried out. The study consisted of three rounds, with response rates ranging from 86.5% to 95%. At each round, experts were asked to assess key elements (defined as the most important elements for the task) and indicators (defined as quantifiable characteristics). At each round, panelists were contacted via e-mail explaining them the tasks to be done and giving them the Web link where to complete the questionnaire. At Round 3, each key element and indicator was assessed as essential, very important, important, accessory, or unnecessary. A 70% agreement was used as cutoff. RESULTS: At Round 3, more than 70% of panelists agreed on six key elements being essential, with one of them (Assuring a good coordination between pediatric and adult professionals) reaching an almost complete consensus (97%). Additionally, 11 more obtained more than 70% agreement when combined with the Very important category. Among indicators, only one (Patient not lost to follow-up) was considered almost unanimously (91%) as essential by the panelists and seven others also reached consensus when the Very important category was included. CONCLUSIONS: Using these results as a framework to develop guidelines at local, national, and international levels would allow better assessing and comparing transition programs.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells.

Successful generation of high producing cell lines requires the generation of cell clones expressing the recombinant protein at high levels and the characterization of the clones' ability to maintain stable expression levels. The use of cis-acting epigenetic regulatory elements that improve this otherwise long and uncertain process has revolutionized recombinant protein production. Here we review and discuss new insights into the molecular mode of action of the matrix attachment regions (MARs) and ubiquitously-acting chromatin opening elements (UCOEs), i.e. cis-acting elements, and how these elements are being used to improve recombinant protein production. These elements can help maintain the chromatin environment of the transgene genomic integration locus in a transcriptionally favorable state, which increases the numbers of positive clones and the transgene expression levels. Moreover, the high producing clones tend to be more stable in long-term cultures even in the absence of selection pressure. Therefore, by increasing the probability of isolating a high producing clone, as well as by increasing transcription efficiency and stability, these elements can significantly reduce the time and cost required for producing large quantities of recombinant proteins.

PERC rule to exclude the diagnosis of pulmonary embolism in emergency low-risk patients: study protocol for the PROPER randomized controlled study.

BACKGROUND: The diagnosis of Pulmonary Embolism (PE) in the emergency department (ED) is crucial. As emergency physicians fear missing this potential life-threatening condition, PE tends to be over-investigated, exposing patients to unnecessary risks and uncertain benefit in terms of outcome. The Pulmonary Embolism Rule-out Criteria (PERC) is an eight-item block of clinical criteria that can identify patients who can safely be discharged from the ED without further investigation for PE. The endorsement of this rule could markedly reduce the number of irradiative imaging studies, ED length of stay, and rate of adverse events resulting from both diagnostic and therapeutic interventions. Several retrospective and prospective studies have shown the safety and benefits of the PERC rule for PE diagnosis in low-risk patients, but the validity of this rule is still controversial. We hypothesize that in European patients with a low gestalt clinical probability and who are PERC-negative, PE can be safely ruled out and the patient discharged without further testing. METHODS/DESIGN: This is a controlled, cluster randomized trial, in 15 centers in France. Each center will be randomized for the sequence of intervention periods: a 6-month intervention period (PERC-based strategy) followed by a 6-month control period (usual care), or in reverse order, with 2 months of "wash-out" between the 2 periods. Adult patients presenting to the ED with a suspicion of PE and a low pre test probability estimated by clinical gestalt will be eligible. The primary outcome is the percentage of failure resulting from the diagnostic strategy, defined as diagnosed venous thromboembolic events at 3-month follow-up, among patients for whom PE has been initially ruled out. DISCUSSION: The PERC rule has the potential to decrease the number of irradiative imaging studies in the ED, and is reported to be safe. However, no randomized study has ever validated the safety of PERC. Furthermore, some studies have challenged the safety of a PERC-based strategy to rule-out PE, especially in Europe where the prevalence of PE diagnosed in the ED is high. The PROPER study should provide high-quality evidence to settle this issue. If it confirms the safety of the PERC rule, physicians will be able to reduce the number of investigations, associated subsequent adverse events, costs, and ED length of stay for patients with a low clinical probability of PE. TRIAL REGISTRATION: NCT02375919 .

Iterative reconstruction in CT : using mathematical model observers to determine low dose images' trustworthiness

La tomodensitométrie (TDM) est une technique d'imagerie pour laquelle l'intérêt n'a cessé de croitre depuis son apparition au début des années 70. De nos jours, l'utilisation de cette technique est devenue incontournable, grâce entre autres à sa capacité à produire des images diagnostiques de haute qualité. Toutefois, et en dépit d'un bénéfice indiscutable sur la prise en charge des patients, l'augmentation importante du nombre d'examens TDM pratiqués soulève des questions sur l'effet potentiellement dangereux des rayonnements ionisants sur la population. Parmi ces effets néfastes, l'induction de cancers liés à l'exposition aux rayonnements ionisants reste l'un des risques majeurs. Afin que le rapport bénéfice-risques reste favorable au patient il est donc nécessaire de s'assurer que la dose délivrée permette de formuler le bon diagnostic tout en évitant d'avoir recours à des images dont la qualité est inutilement élevée. Ce processus d'optimisation, qui est une préoccupation importante pour les patients adultes, doit même devenir une priorité lorsque l'on examine des enfants ou des adolescents, en particulier lors d'études de suivi requérant plusieurs examens tout au long de leur vie. Enfants et jeunes adultes sont en effet beaucoup plus sensibles aux radiations du fait de leur métabolisme plus rapide que celui des adultes. De plus, les probabilités des évènements auxquels ils s'exposent sont également plus grandes du fait de leur plus longue espérance de vie. L'introduction des algorithmes de reconstruction itératifs, conçus pour réduire l'exposition des patients, est certainement l'une des plus grandes avancées en TDM, mais elle s'accompagne de certaines difficultés en ce qui concerne l'évaluation de la qualité des images produites. Le but de ce travail est de mettre en place une stratégie pour investiguer le potentiel des algorithmes itératifs vis-à-vis de la réduction de dose sans pour autant compromettre la qualité du diagnostic. La difficulté de cette tâche réside principalement dans le fait de disposer d'une méthode visant à évaluer la qualité d'image de façon pertinente d'un point de vue clinique. La première étape a consisté à caractériser la qualité d'image lors d'examen musculo-squelettique. Ce travail a été réalisé en étroite collaboration avec des radiologues pour s'assurer un choix pertinent de critères de qualité d'image. Une attention particulière a été portée au bruit et à la résolution des images reconstruites à l'aide d'algorithmes itératifs. L'analyse de ces paramètres a permis aux radiologues d'adapter leurs protocoles grâce à une possible estimation de la perte de qualité d'image liée à la réduction de dose. Notre travail nous a également permis d'investiguer la diminution de la détectabilité à bas contraste associée à une diminution de la dose ; difficulté majeure lorsque l'on pratique un examen dans la région abdominale. Sachant que des alternatives à la façon standard de caractériser la qualité d'image (métriques de l'espace Fourier) devaient être utilisées, nous nous sommes appuyés sur l'utilisation de modèles d'observateurs mathématiques. Nos paramètres expérimentaux ont ensuite permis de déterminer le type de modèle à utiliser. Les modèles idéaux ont été utilisés pour caractériser la qualité d'image lorsque des paramètres purement physiques concernant la détectabilité du signal devaient être estimés alors que les modèles anthropomorphes ont été utilisés dans des contextes cliniques où les résultats devaient être comparés à ceux d'observateurs humain, tirant profit des propriétés de ce type de modèles. Cette étude a confirmé que l'utilisation de modèles d'observateurs permettait d'évaluer la qualité d'image en utilisant une approche basée sur la tâche à effectuer, permettant ainsi d'établir un lien entre les physiciens médicaux et les radiologues. Nous avons également montré que les reconstructions itératives ont le potentiel de réduire la dose sans altérer la qualité du diagnostic. Parmi les différentes reconstructions itératives, celles de type « model-based » sont celles qui offrent le plus grand potentiel d'optimisation, puisque les images produites grâce à cette modalité conduisent à un diagnostic exact même lors d'acquisitions à très basse dose. Ce travail a également permis de clarifier le rôle du physicien médical en TDM: Les métriques standards restent utiles pour évaluer la conformité d'un appareil aux requis légaux, mais l'utilisation de modèles d'observateurs est inévitable pour optimiser les protocoles d'imagerie. -- Computed tomography (CT) is an imaging technique in which interest has been quickly growing since it began to be used in the 1970s. Today, it has become an extensively used modality because of its ability to produce accurate diagnostic images. However, even if a direct benefit to patient healthcare is attributed to CT, the dramatic increase in the number of CT examinations performed has raised concerns about the potential negative effects of ionising radiation on the population. Among those negative effects, one of the major risks remaining is the development of cancers associated with exposure to diagnostic X-ray procedures. In order to ensure that the benefits-risk ratio still remains in favour of the patient, it is necessary to make sure that the delivered dose leads to the proper diagnosis without producing unnecessarily high-quality images. This optimisation scheme is already an important concern for adult patients, but it must become an even greater priority when examinations are performed on children or young adults, in particular with follow-up studies which require several CT procedures over the patient's life. Indeed, children and young adults are more sensitive to radiation due to their faster metabolism. In addition, harmful consequences have a higher probability to occur because of a younger patient's longer life expectancy. The recent introduction of iterative reconstruction algorithms, which were designed to substantially reduce dose, is certainly a major achievement in CT evolution, but it has also created difficulties in the quality assessment of the images produced using those algorithms. The goal of the present work was to propose a strategy to investigate the potential of iterative reconstructions to reduce dose without compromising the ability to answer the diagnostic questions. The major difficulty entails disposing a clinically relevant way to estimate image quality. To ensure the choice of pertinent image quality criteria this work was continuously performed in close collaboration with radiologists. The work began by tackling the way to characterise image quality when dealing with musculo-skeletal examinations. We focused, in particular, on image noise and spatial resolution behaviours when iterative image reconstruction was used. The analyses of the physical parameters allowed radiologists to adapt their image acquisition and reconstruction protocols while knowing what loss of image quality to expect. This work also dealt with the loss of low-contrast detectability associated with dose reduction, something which is a major concern when dealing with patient dose reduction in abdominal investigations. Knowing that alternative ways had to be used to assess image quality rather than classical Fourier-space metrics, we focused on the use of mathematical model observers. Our experimental parameters determined the type of model to use. Ideal model observers were applied to characterise image quality when purely objective results about the signal detectability were researched, whereas anthropomorphic model observers were used in a more clinical context, when the results had to be compared with the eye of a radiologist thus taking advantage of their incorporation of human visual system elements. This work confirmed that the use of model observers makes it possible to assess image quality using a task-based approach, which, in turn, establishes a bridge between medical physicists and radiologists. It also demonstrated that statistical iterative reconstructions have the potential to reduce the delivered dose without impairing the quality of the diagnosis. Among the different types of iterative reconstructions, model-based ones offer the greatest potential, since images produced using this modality can still lead to an accurate diagnosis even when acquired at very low dose. This work has clarified the role of medical physicists when dealing with CT imaging. The use of the standard metrics used in the field of CT imaging remains quite important when dealing with the assessment of unit compliance to legal requirements, but the use of a model observer is the way to go when dealing with the optimisation of the imaging protocols.

No Accumulation of Transposable Elements in Asexual Arthropods.

Transposable elements (TEs) and other repetitive DNA can accumulate in the absence of recombination, a process contributing to the degeneration of Y-chromosomes and other nonrecombining genome portions. A similar accumulation of repetitive DNA is expected for asexually reproducing species, given their entire genome is effectively nonrecombining. We tested this expectation by comparing the whole-genome TE loads of five asexual arthropod lineages and their sexual relatives, including asexual and sexual lineages of crustaceans (Daphnia water fleas), insects (Leptopilina wasps), and mites (Oribatida). Surprisingly, there was no evidence for increased TE load in genomes of asexual as compared to sexual lineages, neither for all classes of repetitive elements combined nor for specific TE families. Our study therefore suggests that nonrecombining genomes do not accumulate TEs like nonrecombining genomic regions of sexual lineages. Even if a slight but undetected increase of TEs were caused by asexual reproduction, it appears to be negligible compared to variance between species caused by processes unrelated to reproductive mode. It remains to be determined if molecular mechanisms underlying genome regulation in asexuals hamper TE activity. Alternatively, the differences in TE dynamics between nonrecombining genomes in asexual lineages versus nonrecombining genome portions in sexual species might stem from selection for benign TEs in asexual lineages because of the lack of genetic conflict between TEs and their hosts and/or because asexual lineages may only arise from sexual ancestors with particularly low TE loads.